Fig. 7. Calpain activity is required for transendothelial migration of DC. After incubation of CFSE-labelled DCs on a monolayer of SVEC 4-10 endothelial cells for 1 hour, co-cultures were washed once with PBS and fixed with 3% paraformaldehyde, permeabilised with 0.05% Triton X-100 and stained to detect actin filaments. (A) Percentage of DC transmigrated, transmigrating or retained on the apical surface of the monolayer. (B) Spread area of DCs under the monolayer in untreated (black), ALLM-treated (grey) and ALLN-treated (white) cells. Values are mean ± s.e.m. of 50 measurements from three coverslips. (C) Skewed confocal micrographs of optical sections at the apical and basal poles of endothelial cell monolayers. CFSE-labelled DCs are green and the distribution of actin filaments is shown in red. Treatment of DCs with calpain inhibitors reduced the transmigration of DCs across the endothelial cells and reduced the spread area under the endothelial cell monolayer. The graphs illustrate the mean percentage of cells ± s.e.m. from three experiments. ^*P<0.05, ^**P<0.01 compared with levels in untreated cells (Student's t-test). The micrographs are representative of the cytoskeletal organisation of DCs detected in three independent experiments.